Abstract

We recently demonstrated that both lisinopril and candesartan, an angiotensin-converting enzyme inhibitor and angiotensin II type 1 receptor blocker, respectively, attenuate pancreatic inflammation and fibrosis in male Wistar Bonn/Kobori (WBN/Kob) rats. The purpose of the present study was to assess whether combination therapy with low doses of both, ineffective when given alone, might synergistically exert protective effects. Lisinopril, candesartan, or a combination of both in drinking water was administered to 10-week-old male WBN/Kob rats for 10 weeks. Parameters of inflammation and fibrosis, positive immunostaining for α-smooth muscle actin, and gene expression of cytokine and growth factors were assessed, as well as circulating renin-angiotensin system components. Dose-dependent effects of combination therapy were also investigated. Only combination therapy attenuated gross alterations in the pancreas, as quantitatively confirmed by increases in pancreatic weights and decreases in myeloperoxidase activity, hydroxyproline content, histologic scores, relative fibrosis area, and relative area of α-smooth muscle actin-positive cells. Combination therapy suppressed up-regulation of tumor necrosis factor-α, platelet-derived growth factor-receptor β, and transforming growth factor-β1 mRNA in the pancreas. Dose dependence of combination therapy was recognized with reference to improvement in these parameters. The conclusions are that combination therapy synergistically alleviated pancreatic inflammation and fibrosis in male WBN/Kob rats. This effect may be related to suppression of tumor necrosis factor-α, platelet-derived growth factor-receptor β, and transforming growth factor-β1 mRNA. Compared with the either therapy alone, combination therapy with an angiotensin-converting enzyme inhibitor and an angiotensin II type 1 receptor blocker may be more beneficial for treating chronic pancreatitis.

The relative paucity of easily reproducible animal models has limited our understanding of the pathogenetic mechanisms responsible for pancreatic inflammation and fibrosis. Male Wistar Bonn/Kobori (WBN/Kob) rats consistently and spontaneously develop chronic pancreatitis, which is accompanied by parenchymal destruction and its replacement with fibrosis, resulting in both endocrine and exocrine dysfunction (Mori et al., 1988). We have demonstrated that acinar cell apoptosis is associated with infiltration of T cells and that tacrolimus, an immunosuppressant, attenuates the apoptosis and chronic inflammation in this model (Yamada et al., 2001). It is well established that T cells infiltrate into the pancreas in human chronic pancreatitis (Okazaki et al., 2000). We thus consider that the present model may be useful for exploring therapeutic strategies for chronic pancreatitis in human, including the autoimmune disease now recognized as a new entity (Okazaki et al., 2000).

There is a growing body of evidence that the renin-angiotensin system (RAS) plays a crucial role in the pathophysiology of fibrosis in the heart, kidney, and liver (Kagami et al., 1994; Bataller et al., 2000; Ostrom et al., 2003). Both angiotensin-converting enzyme (ACE) inhibitors (ACEIs) and angiotensin II (AT-II) type 1 receptor blockers (ARBs) attenuate hepatic fibrosis by suppressing activation of hepatic stellate cells and reducing their production of transforming growth factor (TGF)-β1 in experimental animals, suggesting that AT-II and its type 1 receptor interaction may be intimately involved in hepatic fibrosis (Jonsson et al., 2001; Yoshiji et al., 2001). Furthermore, it has been demonstrated that anti-aldosteronic drugs suppress cardiac and renal fibrosis in experimental animals, so that aldosterone, the last product of the RAS, may also be involved in fibrosis in these organs (Epstein, 2001; Rocha and Funder, 2002).

Pancreatic stellate cells (PSCs), which have some similar properties to their counterparts in liver are involved in the pathogenesis of pancreatic fibrosis in both experimental animals and humans (Apte et al., 1999; Haber et al., 1999). Furthermore, the RAS is present intrinsically in the pancreas, and its gene expression is elevated during acute pancreatitis and chronic pancreatic hypoxia in experimental animals, suggesting an important role of RAS in the pathogenesis of pancreatic injury (Leung et al., 2000; Chan et al., 2000). We recently demonstrated that both lisinopril, an ACEI, and candesartan, an ARB, in drinking water attenuate pancreatic inflammation and fibrosis by suppressing induction of TGF-β1 mRNA and blocking activation of PSCs in male WBN/Kob rats (Kuno et al., 2003; Yamada et al., 2003). However, the effective doses of ACEI and ARB observed in the previous studies are too high for the clinical use.

It is well documented that addition of ARB to ACEI and combination therapy with both drugs can either additively or synergistically exert cardio- and renoprotection in experimental animals and humans (McKelvie et al., 1999; Mogensen et al., 2000; Rossing et al., 2003). These findings strongly suggested the possibility that combination therapy with ACEI and ARB may exert an additive or synergistic effect on pancreatic fibrosis and to test this hypothesis the present study was performed with our WBN/Kob rats. The purposes were to 1) determine whether ineffective doses of lisinopril and candesartan might in combination reduce pancreatic inflammation and fibrosis; 2) assess the effects of combination therapy on circulating components of the RAS and cytokine and growth factors mRNA in the pancreas to clarify mechanisms; and 3) investigate dose dependence of combination therapy in the model.

Materials and Methods

Materials and Animals. Male WBN/Kob and Wistar rats were purchased from SLC (Hamamatsu, Japan) and maintained in a temperature-controlled room under constant light, with free access to water and standard laboratory feed. The study protocol was approved by the Animal Care Committee of Nagoya City University. Lisinopril and candesartan cilexetil (candesartan) were generous gifts from Shionogi Co., Ltd. (Osaka, Japan) and Takeda Co., Ltd. (Osaka, Japan), respectively. All other chemicals were of the highest quality available.

The concentrations of lisinopril and candesartan in drinking water, 20 and 10.5 mg/l, and more than twice of those were earlier found to be ineffective for attenuating pancreatic inflammation and fibrosis in the present model (Kuno et al., 2003; Yamada et al., 2003). The estimated doses in the single therapy groups were 2 and 1 mg/kg/day, respectively (Kuno et al., 2003; Yamada et al., 2003). In the present experiments, lisinopril was dissolved in drinking water, and candesartan was dissolved in 1:1 mix of ethanol and polyethylene glycol, warmed to 60 centigrade, and then 1 N sodium carbonate solution was added before being dissolved in drinking water (Yamada et al., 2003). Untreated group was given water containing equal volume of ethanol, polyethylene glycol, and sodium carbonate solution without lisinopril and candesartan. Fresh solutions were given three times a week, for a total of 10 weeks, and the amount consumed was calculated. Body weights were recorded weekly. Five male Wistar rats (20 week old) were used as nonpancreatitis controls.

Tissue Sampling. All WBN/Kob rats treated for 10 weeks were killed with an overdose of pentobarbital sodium (Abbott Laboratories, North Chicago, IL) after taking blood from the abdominal aorta. Each pancreas was immediately removed and samples were stored at –80 centigrade for determination of myeloperoxidase (MPO) activity and hydroxyproline content, or in liquid nitrogen for eventual determination of cytokine and growth factors mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR). Tissue was also fixed in 10% buffered formalin for histological assessment and immunohistochemistry. Serum and plasma containing ethylenediamine tetraacetic acid sodium were kept at –80 centigrade for eventual determination of ACE (serum), AT-II (plasma), and aldoseterone (serum). In addition, pancreases from 20-week-old Wistar rats were immediately placed in liquid nitrogen for RT-PCR analyses.

Histological Analyses. Pancreas tissues, obtained from both duodenal and splenic lobes, were fixed with 10% buffered formalin and routinely processed for embedding in paraffin, sectioned, and stained with hematoxylin and eosin and Azan. Histological observation was performed using a microscope having the object lens of 10×, intermittent lens of 4×, and eyepiece lens of 10× (Olympus BH-2, Olympus Co., Tokyo, Japan). The histological status of inflammation in each animal was evaluated by a pathologist who was unaware of the groups, with reference to the grades of inflammatory cell infiltration, interstitial edema, acinar cell necrosis, hemorrhage, and fibrosis, as reported previously (Yamada et al., 2001, 2003; Kuno et al., 2003). Each factor was graded as none, mild, moderate, and severe, scored as 0, 1, 2, and 3, respectively. Quantity of fibrosis was analyzed under the microscope (Olympus BH-2, Olympus Co., Tokyo, Japan) connected with the 3CCD color video camera (DXC-950, Sony Co., Tokyo, Japan) with the aid of an image processor (Image Processor for Analytical Pathology; Sumica Technoservise, Osaka, Japan). The percentage of aniline-blue positive fibrous tissue per total area in whole Azan-stained pancreas section was measured except lymph nodes and major vessels, if included (Kuno et al., 2003; Yamada et al., 2003). The results were then used to calculate values relative to the control cases, set at 100%.

Immunohistochemistry for α-Smooth Muscle Actin. Tissues were stained with mouse anti-human α-smooth muscle actin (α-SMA) monoclonal antibody (DakoCytomation California Inc., Carpiteria, CA), which can detect rat α-SMA as described previously (Kuno et al., 2003; Yamada et al., 2003). The relative area of α-SMA-positive cells per total area was also evaluated as described for “Histological Analyses”.

AT-II in the plasma containing ethylenediamine tetraacetic acid sodium was determined by radioimmunoassay as reported previously (Iwahana et al., 1996). Briefly, plasma was incubated with a rabbit anti-AT-II antibody and 125I-labeled AT-II and then a goat antibody for rabbit immunoglobulin. After adding polyethylene glycol, the radioactivity of the pellet was measured using a counter. The amount of AT-II was calculated using a standard curve generated using angiotensin II human.

Serum aldosterone was determined with a commercially available aldosterone radioimmunoassay kit II (Dinabott Co., Tokyo, Japan) and expressed as picograms per milliliter.

The relative expression intensity of each mRNA band was determined with an image analyzer (NIH Image), and semiquantitative analyses relative to β-actin were preformed.

Statistics. Data are expressed as arithmetic means ± standard deviations. Statistical differences among groups were identified using the one-way analysis of variance, followed by multiple comparisons using the least significance difference method. The Mann-Whitney's U test was used for statistical analyses of the histologic scores, relative fibrosis area, and relative area of α-SMA-positive cells.

Results

Doses of Lisinopril and Candesartan, Body Weights, and Pancreas Weights. The average doses of lisinopril and candesartan in the single therapy groups calculated from water consumption and body weights were 2.34 ± 0.18 and 0.94 ± 0.05 mg/kg/day, respectively, during the experimental period. Those of lisinopril and candesartan in the combination group were 2.40 ± 0.07 and 1.26 ± 0.04 mg/kg/day, respectively. Body weights were significantly lower in the combination group compared with the other groups (Table 2). The pancreas weights were significantly higher in the combination group than in the untreated and candesartan groups.

Effects of lisinopril, candesartan, and combination of lisinopril and candesartan in drinking water on body weight, pancreas weight, pancreas MPO activity, and pancreas hydroxyproline content in 20-week-old male WBN/Kob rats

Statistical analyses were performed with one-way analysis of variance, followed by multiple comparisons using the least significance difference method.

Macroscopic Findings. The pancreases in the untreated, lisinopril, and candesartan groups had widespread brown and red foci (Fig. 1, A–C, respectively). In sharp contrast, the pancreases in the combination group had almost intact appearances (Fig. 1D) as observed in the pancreas in Wistar rat (not shown).

Histological Analyses. Focal severe inflammation in the pancreas was evident in the untreated group (Fig. 2A). This was characterized by neutrophil and lymphocyte infiltration, interstitial edema, hemorrhage, and occasional acinar cell necrosis. Fibrosis was noted as replacing acini, being also found between remaining acini. Neither lisinopril nor candesartan had any significant effects on these inflammatory changes (Fig. 2, B and C). However, the combination therapy dramatically reduced the inflammatory changes, resulting in limited foci of lymphocyte infiltration with slight fibrosis (Fig. 2D). No histological alteration in the pancreas was present in Wistar rat (Fig. 2E). Significant reduction of inflammatory scores was observed for almost all parameters in the combination group compared with the untreated, lisinopril, and candesartan groups (Table 3). Relative fibrosis area calculated by Azan-stained area in the pancreas tended to be decreased to 55.6 ± 19.0% in the combination group compared with 100% set for the untreated group.

Effects of lisinopril, candesartan, and combination of lisinopril and candesartan in drinking water on histologic scores, relative fibrosis area, and relative area of α-smooth muscle actin-positive cells of the pancreas in 20-week-old male WBN/Kob rats

Scores for each factor are averages and SDs for the grade, none, mild, moderate, and severe (0-3). Statistical analyses were performed with Mann-Whitney U test.

Immunohistochemistry for α-Smooth Muscle Actin. α-SMA-positive cells with the morphology of activated PSCs were localized in the peri-acinar fibrotic areas and vascular walls in the untreated, lisinopril, and candesartan groups (not shown). In contrast, α-SMA-positive cells were observed only in the vascular walls in the combination group. Decrease in the relative area of α-SMA-positive cells was noted, although it did not reach statistical significance (Table 3).

Pancreatic Myeloperoxidase Activity. Pancreatic MPO activity, an indirect index of granulocyte infiltration, was significantly suppressed in the combination group compared with those in the untreated and candesartan groups (Table 2).

Serum Angiotensin-Converting Enzyme Activity, Plasma Angiotensin II Levels, and Serum Aldosterone Levels. Serum ACE activity and plasma AT-II levels were significantly decreased in the lisinopril and combination groups compared with those in untreated and candesartan groups, respectively. Serum aldosterone levels were not significantly decreased in the combination group compared with those of other groups (Table 4).

Effects of lisinopril, candesartan, and combination of lisinopril and candesartan in drinking water on serum ACE activity, plasma angiotensin II levels, and serum aldosterone levels in 20-week-old male WBN/Kob rats

Statistical analyses were performed with one-way analysis of variance, followed by multiple comparisons using the least significance difference method.

Expression of Tumor Necrosis Factor-α, Platelet-Derived Growth Factor-B, Platelet-Derived Growth Factor-Receptor β, and Transforming Growth Factor-β1 mRNA. RT-PCR revealed TNF-α, PDGF-Rβ, and TGF-β1 mRNA to be overexpressed in the pancreas in the untreated group, whereas they were detected at only low levels in male Wistar rats (Fig. 3). However, PDGF-B mRNA was not up-regulated in the untreated groups compared with that in male Wistar rats. Although lisinopril and candesartan alone did not alter TNF-α, PDGF-Rβ, and TGF-β1 mRNA levels, the combination therapy suppressed up-regulation of all three mRNAs. Semiquantitative analysis of each mRNA relative to β-actin revealed that TNF-α, PDGF-R β, and TGF-β1 mRNA were significantly up-regulated in the untreated group compared with Wistar rats and they were suppressed in the combination group (Table 5).

Expression of TNF-α, PDGF-B, PDGF-Rβ, and TGF-β1 mRNAs analyzed by RT-PCR. Findings are for untreated, lisinopril, candesartan, and combination groups and 20-week-old male Wistar rats. The β-actin is included in each pancreas tissue as an internal control.

Statistical analyses were performed with one-way analysis of variance, followed by multiple comparisons using the least significance difference method.

The relative expression intensity of each band was determined with an image analyzer and semiquantitative analyses relative to β-actin were performed.

Doses of Lisinopril and Candesartan, Body Weights, and Pancreas Weights in the Dose-Dependence Study. Next, the dose-dependent effects of the combination therapy were assessed. The average doses of lisinopril in the low and medium combination and combination groups were 0.29 ± 0.01, 0.62 ± 0.06, and 2.27 ± 0.12 mg/kg/day, respectively. Those of candesartan were 0.41 ± 0.01, 0.87 ± 0.08, and 1.19 ± 0.06 mg/kg/day, respectively. The body weights were significantly lower in all three combination groups than in the untreated group, and the pancreas weights were significantly higher (Table 6).

Statistical analyses were performed with one-way analysis of variance, followed by multiple comparisons using the least significance difference method.

Dose Dependence of the Combination Therapy Effects on Histological Findings. In line with earlier studies, untreated animals showed severe inflammation and fibrosis in the pancreas (Fig. 4A). Limited foci of severe and moderate focal inflammation remained in the low and medium combination groups, respectively (Fig. 4, B and C), so that protective effects assessed by histologic scores were observed with regard to acinar cell necrosis in the low combination group and acinar cell necrosis and fibrosis in the medium combination group, respectively (Table 7). In contrast, significant reduction was noted for all histologic scores in the combination group (Fig. 4D; Table 7). The relative fibrosis areas were significantly decreased in all three combination groups compared with the untreated group. The relative area of α-SMA-positive cells was significantly decreased in the medium combination group and tended to be decreased in the low combination and combination groups (Table 7).

Effects of low and medium combination and combination of lisinopril candesartan in drinking water on histologic scores, relative fibrosis area, and relative area of α-smooth muscle actin-positive cells of the pancreas in 20-week-old male WBN/Kob rats

Scores for each factor are averages and SDs for the grade, none, mild, moderate, and severe (0-3). Statistical analyses were performed with Mann-Whitney U test.

Dose Dependence of the Combination Therapy Effects on Pancreatic Myeloperoxidase Activity and Pancreatic Hydroxyproline Content. Pancreatic MPO activity and pancreatic hydroxyproline content were significantly decreased in the three combination groups compared with those in the untreated group (Table 6).

Discussion

The present study for the first time demonstrated synergistic preventive effects of combination therapy with ACEI and ARB on chronic inflammation and fibrosis in a visceral organ, the pancreas. The combination therapy with lisinopril and candesartan, ineffective when given alone, attenuated pancreatic inflammation and fibrosis in male WBN/Kob rats. This was quantitatively reflected in increased pancreatic weights and decreased MPO activity (an index of granulocyte infiltration), hydroxyproline content (an index of collagen deposition), histologic scores, relative area of α-SMA-positive cells (an index of stellate cell activation), and relative fibrosis area. Furthermore, the combination therapy suppressed up-regulation of TNF-α, PDGF-Rβ, and TGF-β1 mRNAs in the pancreas. We speculate that suppression of TNF-α mRNA may lead to decreased production of cytokine and reduced inflammatory response. Furthermore, suppression of PDGF-Rβ and TGF-β1 mRNA might account for prevention of pancreatic stellate cell activation and pancreatic fibrosis. We additionally demonstrated dose dependence of the protective effects of combination therapy.

We recently reported that lisinopril, an ACEI, and candesartan, an ARB, in drinking water suppress pancreatic inflammation and fibrosis in the present model (Kuno et al., 2003; Yamada et al., 2003). However, the doses were much higher than the maximal clinical doses (lisinopril, 21.3 mg/kg/day, 65-fold; and candesartan, 13.9 mg/kg/day, 70-fold). Since ACEI and ARB suppress the RAS by pharmacologically different mechanisms, the combination therapy with ACEI and ARB might be expected to be safer and more effective in inhibiting AT-II function than single therapy with twice doses of either agent. It was well documented that the combination therapy with ACEI and ARB has advantages over either therapy alone at low doses for treating heart and kidney diseases in the experimental models and humans (McKelvie et al., 1999; Mogensen et al., 2000; Nakamura et al., 2003; Rossing et al., 2003). These findings prompted us to explore the effect of the combination therapy on chronic pancreatitis. Since the single therapies with more than twice of the present single doses were reported to be ineffective for treating chronic pancreatitis (lisinopril, 5.1 ± 0.5 mg/kg/day, Kuno et al., 2003; candesartan, 4.3 ± 0.3 mg/kg/day, Yamada et al., 2003), we defined the protective effect of the combination therapy as synergistic.

The serum levels of lisinopril and candesartan in the combination therapy are estimated to be around 18 and 55 ng/ml, respectively, from the data of the previous studies (Kuno et al., 2003; Yamada et al., 2003). Since pancreatic weights were increased and pancreatic MPO activity, pancreatic hydroxyproline content, and some histologic scores were decreased in the low and medium combination groups compared with the untreated group in the study of dose dependence, the combination therapy with lower doses of both drugs could still exert protective effects. Considering that the calculated doses of lisinopril and candesartan were 0.29 ± 0.01 and 0.41 ± 0.01 mg/kg/day in the low combination group, it is possible that combination therapy with the maximal clinical doses of both drugs (lisinopril, 0.33 mg/kg/day and candesartan, 0.20 mg/kg/day) may alleviate pancreatic inflammation and fibrosis in cases of human chronic pancreatitis.

Since both lisinopril and candesartan are widely used as antihypertensive drugs in humans, one might speculate that they may decrease blood pressure in the present model. However, the combination therapy with much higher doses of ACEI and ARB did not cause significant decrease in blood pressure in rats with myocardial infarction (Nakamura et al., 2003). It has been well documented that the combination therapy with ACEI and ARB did not cause excessive decrease in blood pressure in human (McKelvie et al., 1999; Mogensen et al., 2000; Rossing et al., 2003). Together, it was speculated that the combination therapy may not cause excessive decrease in blood pressure in the present study.

TNF-α, a proinflammatory cytokine, is expressed in acinar cells and infiltrating macrophages in acute pancreatitis (Gukovskaya et al., 1997). It is reported that TNF-α released from acinar cells is involved in the development of pancreatitis by mediating early inflammatory reactions (Gukovskaya et al., 1997; Xie et al., 2001a). Its inhibition reduces acute pancreatic damage and improves survival with acute pancreatitis in rats (Hughes et al., 1996). Moreover, tissue expression of TNF-α is involved in the onset of chronic pancreatitis in the present model (Xie et al., 2001b). Thus, we assessed tissue TNF-α mRNA levels and confirmed up-regulation in the untreated group. Only combination therapy suppressed this.

PDGF, a dimerized protein secreted by infiltrated inflammatory cells, including macrophages and platelets, induces proliferation of PSCs (Apte et al., 1999; Luttenberger et al., 2000). It consists of two polypeptide chains linked via disulfide bonds and has three isoforms, PDGF-A, PDGF-B, and PDGF-AB, with PDGF-B having the most potent effects in vitro (Apte et al., 1999). The PDGF receptor is composed of two types, α and β, which recognize all three types of PDGF and PDGF-B, respectively. PDGF-B and PDGF-Rβ, but not PDGF-Rα, are closely associated with fibrosis in human and experimental pancreatitis (Ebert et al., 1998; Haber et al., 1999). Furthermore, PDGF-B mRNA is up-regulated in dibutyltin dichloride-induced chronic pancreatitis (Inoue et al., 2002). We here found PDGF-Rβ mRNA to be up-regulated in the present model, whereas PDGF-B mRNA was unchanged. Again, only combination therapy suppressed this up-regulation of PDGF-Rβ mRNA.

AT-II directly induces proliferation of pancreatic stellate cells as well as digestive enzyme secretion by acinar cells in vitro, both of which were abolished in the presence of ARBs (Reinehr et al., 2004; Hama et al., 2004; Tsang et al., 2004). We speculate that inhibition of AT-II by the combination therapy may have similar effects in the present model. Moreover, AT-II is a potent vasoconstrictor, and ACEIs increase vasodilatory factors, including bradykinin, nitric oxide, and prostaglandin (Campbell et al., 1994). The combination therapy could thus increase pancreatic blood flow by both blocking AT-II function and increasing vasodilatory factors, thereby improving pancreatic ischemia, which was reported to be one element in the pathogenesis of chronic pancreatitis (Mori et al., 1988). Moreover, interaction between AT-II and its type 1 receptor may result in secretion of aldosterone, which may be involved in cardiac and renal fibrosis in the experimental animals (Epstein, 2001; Rocha and Funder, 2002). In fact, combination therapy with an ACEI and an ARB resulted in greater decrease in circulating aldosterone levels than the either agent alone in the RESOLVD study (McKelvie et al., 1999). To our knowledge, no reports are available regarding the role of aldosterone in chronic pancreatitis. We hypothesized that protection might be related to its decrease, but contrary to expectation, the combination therapy did not cause further depress of serum aldosterone levels compared with single lisinopril therapy, suggesting that aldosterone may not be involved in the pathogenesis of pancreatic fibrosis.

There are sporadic reports demonstrating that ACEIs caused acute pancreatitis in humans (Madsen and Jacobsen, 1995; Muchnick and Mehta, 1999). Although we were able to reduce the effective dose of lisinopril by combined administration with candesartan, caution may be necessary when lisinopril is used for treating chronic pancreatitis in humans.

In conclusion, the combination therapy with lisinopril and candesartan may synergistically alleviate chronic pancreatic inflammation and fibrosis in male WBN/Kob rats by suppressing up-regulation of TNF-α, PDGF-Rβ, and TGF-β1 mRNA, resulting in the prevention of pancreatic stellate cell activation and pancreatic fibrosis. We propose that combination therapy with an ACEI and an ARB may allow reduction of effective doses so that treatment of chronic pancreatitis with such agents will become possible. Further studies in this area are clearly warranted.